Increased susceptibility of aging kidney to ischemic injury: identification of candidate genes changed during aging, but corrected by caloric restriction

Abstract

Aging is associated with an increased incidence and severity of acute renal failure. However, the molecular mechanism underlying the increased susceptibility to injury remains undefined. These experiments were designed to investigate the influence of age on the response of the kidney to ischemic injury and to identify candidate genes that may mediate this response. Renal slices prepared from young (5 mo), aged ad libitum (aged-AL; 24 mo), and aged caloric-restricted (aged-CR; 24 mo) male Fischer 344 rats were subjected to ischemic stress (100% N(2)) for 0-60 min. As assessed by biochemical and histological evaluation, slices from aged-AL rats were more susceptible to injury than young counterparts. Importantly, caloric restriction attenuated the increased susceptibility to injury. In an attempt to identify the molecular pathway(s) underlying this response, microarray analysis was performed on tissue harvested from the same animals used for the viability experiments. RNA was isolated and the corresponding cDNA was hybridized to CodeLink Rat Whole Genome Bioarray slides. Subsequent gene expression analysis was performed using GeneSpring software. Using two-sample t-tests and a twofold cut-off, the expression of 92 genes was changed during aging and attenuated by caloric restriction, including claudin-7, kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7). Claudin-7 gene expression peaked at 18 mo; however, increased protein expression in certain tubular epithelial cells was seen at 24 mo. Kim-1 gene expression was not elevated at 8 or 12 mo but was at 18 and 24 mo. However, changes in Kim-1 protein expression were only seen at 24 mo and corresponded to increased urinary levels. Importantly, these changes were attenuated by caloric restriction. MMP-7 gene expression was decreased at 8 mo, but an age-dependent increase was seen at 24 mo. Increased MMP-7 protein expression in tubular epithelial cells at 24 mo was correlated with the gene expression pattern. In summary, we identified genes changed by aging and changes attenuated by caloric restriction. This will facilitate investigation into the molecular mechanism mediating the age-related increase in susceptibility to injury.

Fig. 1

Schematic depiction of the process used to filter and analyze the microarray data.

Fig. 2

Impact of ischemia on the viability of renal tissue slices. A: kidney slices were harvested from young, aged ad libitum (aged-AL), or aged caloric-restricted (aged-CR) rats and challenged by simulated ischemia (100% N₂) for 30 or 60 min. Viability was assessed by intracellular ATP and GSH content or leakage of LDH and αGST into the culture media. The results were compared with control slices (cultured in 95:5 O₂-CO₂) from each respective group. Each data point represents means ± SD for 4 animals (4 slices per animal). *Significant difference between young and aged-AL. **Significant difference in aged-AL compared with young and aged-CR. B: following 60 min, slices were harvested and processed for histological evaluation. Normal tubular structure is seen in control slices from young, aged-AL, and aged-CR rats; however, anoxia is associated with significant damage to tubules including areas of flattened tubular epithelium, cell vacuolization, reduced eosin staining, suggesting loss of cytoplasmic proteins and cell sloughing in aged-AL but not young and aged-CR rats. The width of the field is 870 µm.

Fig. 3

Impact of aging on renal gene expression. A: normalized Claudin-7 (Cldn-7), kidney injury molecule-1 (Kim-1), and matrix metalloproteinase-7 (MMP-7) gene expression in young, aged-AL, and aged-CR rats as assessed by microarray analysis using the CodeLink Rat Whole Genome Bioarray. Each data point represents the normalized mean intensity ± SD for that gene across all arrays (4 animals). **Significant difference in aged-AL compared with young and aged-CR. B: age-related changes in the above target genes were verified by quantitative PCR. The β-actin normalized Cldn-7, Kim-1, and MMP-7 gene expression in young, aged-AL, and aged-CR rats is presented relative to the gene expression in an arbitrary reference sample (Stratagene Rat Universal Reference RNA). The values represent means ± SD of relative gene expression of 8 animals per group. **Significant difference in aged-AL compared with young and aged-CR.

Fig. 4

Impact of aging on claudin-7 expression. A: claudin-7 gene expression was assessed using quantitative PCR; the β-actin normalized claudin-7 gene expression is presented relative to the gene expression in an arbitrary reference sample (Stratagene Rat Universal Reference RNA). The values represent means ± SD of relative gene expression of 4 animals per group. **Significant difference in aged-AL compared with young and aged-CR. B: Western blot analysis of claudin-7 protein levels in kidney lysates. Full-length claudin-7 is seen at ~25 kDa; membranes were stripped and reprobed with an antibody against β-actin to demonstrate equal loading. C: paraffin-embedded sections were processed for immunohistochemical localization of claudin-7 with a commercially available system. The arrows point to tubules with increased intensity of claudin-7 staining; similar results were seen in duplicate experiments.

Fig. 5

Impact of aging on Kim-1 expression. A: quantiative PCR analysis of Kim-1 gene expression; the β-actin normalized Kim-1 gene expression is presented relative to the gene expression in an arbitrary reference sample (Stratagene Rat Universal Reference RNA). The values represent means ± SD of relative gene expression of 4 animals per group. *Significant difference in 24-mo AL compared with young. **Significant difference in aged-AL compared with young and aged-CR. B: Western blot analysis of Kim-1 protein levels in kidney lysates. Full-length Kim-1 is seen at ~80 kDa, while fragments at 30 and 45 kDa are also seen; the bands at 50 kDa are nonspecific but demonstrate equal protein loading. C: paraffin-embedded sections were processed for immunohistochemical localization of Kim-1 with a commercially available system (Zymed); similar results were seen in duplicate experiments. D: Kim-1 levels in rat urine as assessed by ELISA. Each data point represents means ± SD of 4 animals per group. **Significant difference in 24-mo AL compared with young and 24-mo CR.

Fig. 6

Impact of aging on MMP-7 expression. A: MMP-7 gene expression was assessed using quantitative PCR; the β-actin-normalized MMP-7 gene expression is presented relative to the gene expression in an arbitrary reference sample (Stratagene Rat Universal Reference RNA). The values represent means ± SD of relative gene expression of 4 animals per group. *Significant difference in 24-mo AL compared with young. **Significant difference in aged-AL compared with young and aged-CR. B: Western blot analysis of MMP-7 protein levels in kidney lysates. Full-length MMP-7 is seen at ~27 kDa; membranes were stripped and reprobed with an antibody against β-actin to demonstrate equal loading. C: paraffin-embedded sections were processed for immunohistochemical localization of MMP-7 with a commercially available system; similar results were seen in duplicate experiments.